Physiological Characterization of Lipid Assimilation

To determine the growth on different Tween varieties and with fatty acids, the strains were first grown on mDixon at 33°C for 7 days. The fungal cells were suspended in 3 ml of water with 0.1% Tween 80 used to inoculate 27 mL of minimal medium (MM) [containing per liter: 10 mL K-buffer pH 7.0 (200 g L^-1 K₂HPO₄; Sigma–Aldrich, United States), 145 g L^-1 KH₂PO₄ (Sigma–Aldrich, United States), 20 mL M-N (30 g L^-1 MgSO₄.7H₂O; Sigma–Aldrich, United States), 15 g L^-1 NaCl (Sigma–Aldrich, United States), 1 mL 1% CaCl₂.2H₂O (Sigma–Aldrich, United States) (w/v), 10 mL 20% glucose (Sigma–Aldrich, United States) (w/v), 10 mL 0.01% FeSO₄ (Sigma–Aldrich, United States) (w/v), 5 mL spore elements (100 mg L^-1 ZnSO₄.7H₂O; Sigma–Aldrich, United States), 100 mg L^-1 CuSO₄.5H₂O (Sigma–Aldrich, United States), 100 mg L^-1 H₃BO₃ (Sigma–Aldrich, United States), 100 mg L^-1 MnSO₄.H₂O (Sigma–Aldrich, United States), l00 mg L^-1 Na₂MoO₄.2H₂O (Sigma–Aldrich, United States), and 2.5 mL 20% NH₄NO₃ (Sigma–Aldrich, United States) (w/v)] containing 4 mM Tween [20, 40, 60 or 80 (Sigma–Aldrich, United States)], or 4 mM oleic acid (Carlo Erba), or palmitic acid (Merck) supplemented with 1% Brij-58 (Sigma–Aldrich, United States), an emulsifier that is not metabolized. It did not support the growth of the FAS mutant of the yeast S. cerevisiae (Schweizer and Bolling, 1970) that was grown for 3 days at 33°C. Subsequently, 0.3 mL was used to inoculate 29.7 mL of fresh MM containing either 4 mM Tween 20, 40, 60, 80, oleic acid, and/or palmitic acid in 1% Brij-58, with mDixon broth as the positive control. Growth was followed during 8 days in the medium containing Tween 40, palmitic acid, oleic acid, or mixtures of palmitic and oleic acid by determining the colony-forming unit (CFU) by plating on mDixon plates with subsequent incubation at 33°C. In the medium containing Tweens and oleic acid, the optical density (OD) at 600 nm was measured and plating aliquots of the liquid cultures on mDixon plates at 33°C was used to determine the viability of the cells after 8 days of growth. The fatty acids used for culturing were analyzed for composition via a gas chromatography–flame ionization detector; separation was reached using an RTX-Wax column (30 m × 0.25 mm × 0.5 μm) of RESTEK^®. FAMEs were identified by comparing their retention times with those identified with a Supelco^® 37 Component FAME Mix standard. Quantification was intended as a relative concentration. Palmitic acid (Merck) contained 98% palmitic acid and 2% elaidic acid, an unsaturated acid. The oleic acid (Carlo Erba) contained 78% oleic acid and, in addition, we detected polyunsaturated fatty acids (10% of linoleic acid), unsaturated fatty acids (3% palmitoleic acid and 2% elaidic acid), and saturated fatty acids (6% of palmitic acid and 1% heptadecanoic acid).